1. Field of the Invention
The present invention relates generally to the field of physiology and molecular biology. More specifically, the present invention relates to DNA damage and the effects of DNA damage on atherosclerosis.
2. Description of the Related Art
Reactive oxygen species (reactive oxygen species) have been suggested to play a critical role in the pathogenesis of atherosclerotic lesions (1-6), but the underlying mechanisms have not yet been elucidated. For example, reactive oxygen species-mediated mechanisms are likely to be a significant factor in the oxidation of LDL (ox-LDL), a key event in atherogenesis (3,7,8). Studies have shown that both superoxide (O231 ) and peroxynitrite (peroxynitrite; formed from O2−+ nitric oxide) are capable of oxidizing LDL (9-11). Hence, reactions involving nitric oxide and/or O2− are believed to play a critical role in the pathogenesis of atherosclerotic lesions and impaired vascular function (i.e. endothelial cell dysfunction), with the actions of their oxidizing products (H2O2, peroxynitrite) not yet well defined.
The mitochondrion is a major source of cellular reactive oxygen species (O2−), which are formed during electron transport (12-16). These reactive oxygen species are capable of preferentially damaging the mitochondrial membranes and proteins (17-19), affecting key cell functions, including mitochondrial respiration, which, if altered, leads to increased reactive oxygen species production (20-22), mediating lipid peroxidation (23, 24) and DNA damage (25, 26). Because mitochondrial oxidative phosphorylation (OXPHOS) capacities decline as mitochondrial DNA (mtDNA) damage and mutations accumulate with age (6, 27-29), mitochondrial damage and reactive oxygen species generation may act as catalysts for age-related degenerative disease, such as coronary artery disease (CAD). It was hypothesized that free radicals generated within the endothelial and smooth muscle cell environment mediate mitochondrial damage within these cells, establishing a vicious cycle of further reactive oxygen species generation and mitochondrial damage leading to vascular cell dysfunction.
Coronary atherosclerotic heart disease is the leading cause of death in the Western world. Although there is considerable controversy about the exact sequence of events leading to coronary atherosclerotic heart, there is growing evidence that atherosclerotic lesions result from factors mediated by reactive oxygen species. Macrophages recognize and internalize ox-LDL via “scavenger” receptors, becoming foam cells. Accumulation of these foam cells is associated with long-term changes in vascular physiology, including smooth muscle cell migration and proliferation, synthesis of extracellular matrix proteins, and further endothelial cell dysfunction, all core components of atherosclerotic plaques. Similarly, many of the risk factors for coronary atherosclerotic heart are related to increased reactive oxygen species production (i.e. smoking and hypercholestermia). Within the artery, reactive oxygen species can be induced by metabolic processes (mitochondrial oxidative phosphorylation), cytokine or growth factor activation, macrophage or neutrophil stimulation (inflammatory response), and the reaction of nitric oxide with superoxide to yield peroxynitrite, which in turn, generates singlet oxygen and hydroxyl radicals. Hence, while there are a variety of processes that are important for atherogenesis, reactive oxygen species-mediated mechanisms and their effects are among the most significant.
Numerous studies have implicated the mitochondria as a vulnerable target for reactive oxygen species. The association of the mitochondrial DNA with the matrix side of the inner membrane makes it susceptible to membrane disturbances, and a potential target for electrophiles generated in the membrane. Aside from its close association with the inner membrane and OXPHOS, additional factors which make the mitochondrial DNA sensitive to damage are the lack of protective histone and non-histone proteins, and its limited DNA repair capacity. Previous studies have shown that the mitochondria are susceptible to reactive oxygen species mediated damage, manifested in extensive lipid peroxidation and mitochondrial DNA damage. Specifically, it has been shown that reactive oxygen species treatment of endothelial cells results in preferential mitochondrial DNA damage, decreased mitochondrial DNA transcripts, and mitochondrial OXPHOS dysfunction.
The prior art is deficient in methods of measuring oxidative stress that contributes to atherogenesis. The present invention fulfills this long-standing need and desire in the art.